1. Field of the Invention
The present invention relates to four novel compositions of Nigella sativa that function uniquely to reduce oxidative stress, inhibit secretion of prostate specific antigen (PSA) from prostate cells, uncouple mitochondrial membrane potential, inhibit inducible nitric oxide synthase (iNOS) in inflamed fat and muscle tissue, modify fatty acid flux, activate myocyte AMP-activated protein Kinase (AMPK), and inhibit loss of transepithelial electrical resistance (TEER) in stressed intestinal epithelial cells in a manner that is unexpectedly both quantitatively and qualitatively superior to thymoquinone (TQ), the putative active phytochemical of N. sativa (FIG. 1). These compositions would be useful in the prevention or treatment of metabolic disorders such as adaptive thermogenesis, obesity, diabetes, and metabolic syndrome as well as hyperlipidemia, hypertension and exercise recovery. The compositions would also be useful in the treatment or amelioration of benign prostate hyperplasia.
2. Description of the Related Art
Nigella sativa, commonly known as black seed or black curcumin, is traditionally used in the Indian subcontinent, Arabian countries, and Europe for culinary and medicinal purposes as a natural remedy for a number of illnesses and conditions that include asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and influenza. Much of the biological activity of the seeds is believed to be due to TQ, a component of the essential oil, which is also present in the fixed oil.
The seeds of N. sativa as well as TQ are characterized by a very low degree of toxicity. Administration of either the seed, its extract or its oil has been shown not to induce significant toxicity or adverse effects on liver or kidney functions even at extremely high doses [Ali B H, Blunden G. 2003. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 17: 299-305]. Thus, N. sativa seed and TQ possess the necessary safety factor for commercialization in the dietary supplement or pharmaceutical market.
TABLE 1Chemical Content of Various Oil Fractions of N. sativa SeedsCONTENT [%SEED FRACTIONCOMPONENT[w/w]Fixed Oils36Non-fat componentsFatty acids, protein, thiamin,58ribovflavin, pyridoxine, niacin,folic acid, and calcium.Essential fatty acidsMyristic acid (C14)0.5in fixed oilPalmitic acid (C16)13.7Palmitoleic acid (C16 ω-9)0.1Stearic acid (C18)2.6Linoleic acid (C18 ω-6)57.9Linolenic acid (C18 ω-30.2Arachidic acid (C20)1.3Essential oil components (0.5-1.5%): α-Pinene, camphene, β-pinene, sabinene, β-myrcene, α-terpinene, limonene, β-phellandrene, 1,8-cineole, γ-terpinene, p-cymene (7.1-15.5%), α-terpinolene, 2-heptanal, thujone, trans-sabinenehydrate, longipinene, camphor, linalool, cis-Sabinenehydrate, longifoline (1.0-8.0%), bornylacetate, 2-undecanone, 4-terpineol (2.0-6.6%), borneol, carvone, thymoquinone (27-57%), 2-tridecanone, t-anethole (0.25-2.3%), p-cymene-8-ol, p-anisaldehyde, thymol and carvacrol (5.8-11.6%) (Burits, M.; Bucar, F., Antioxidant activity of Nigella sativa essential oil. Phytother Res 2000, 14 (5), 323-8).
As seen in Table 1, the TQ or dithymoquinone content of the essential (volatile) oil fraction is roughly 27-57 percent. The essential oil fraction, however, constitutes only one percent of the seed oils. Thus, TQ comprises only about 0.3 to 0.6% of the fixed oil fraction, the most common commercially available product of N. sativa seeds.
Extraction Processes—
Traditional solvent extraction is time-consuming, requires multiple steps, and consumes large amounts of organic solvents. The amount and the price of organic solvent directly influences the total cost of producing an acceptable extract or product. Moreover, when the final product is used as a food ingredient, it is absolutely necessary to remove all potentially toxic solvents.
Supercritical fluid extraction (SFE) has already proven itself as an attractive technique for selectively removing compounds from complex food matrices. Extraction with liquid or supercritical CO2 is essentially a simple concept, although specialized equipment and technically skilled operators are needed to bring concept to reality. CO2 can exist in solid, liquid or gaseous phase, in common with all chemical substances. Furthermore, if the liquid phase is taken beyond the so-called critical points of temperature and pressure, a supercritical fluid is formed, which in simple terms can be considered as a dense gas1. Both liquid and supercritical CO2 act effectively as solvents. While liquid CO2 is excellent for dissolving relatively non-polar, small molecules (liquid CO2 can be compared to hexane in this regard), supercritical CO2 allows the extraction of larger and more polar compounds. Thus, supercritical extraction has the potential for creating novel extracts of commonly used herbs.
Supercritical CO2 is pumped through the plant material in the extraction columns, where extraction of the desired plant components takes place. After passing through the expansion valve, the extract-laden CO2 is depressurised and the extract precipitates out of solution in the separator. The gaseous CO2 can be recycled for further extractions (FIG. 2).
What sets liquid and supercritical CO2 apart from other solvents such as hexane and ethanol are two key properties. Firstly, once the extraction has been effected, the CO2 solvent is released as a gas and recycled in the process, so that a solvent-free extract is produce. This has two immediate benefits—the extract is free of all solvent residues, and importantly so is the extracted material, which can then be further used for processing if required. Secondly, the solvating power of CO2 can be manipulated readily by altering temperature and pressure. This means that extraction can be highly selective and novel.
Obesity is a disease resulting from a prolonged positive imbalance between energy intake and energy expenditure. In 2000, an estimated 30.5% of adults in the U.S. were obese (i.e. had a body mass index [BMI] greater than 30 kg/m2) and 15.5% of adolescents were overweight (BMI of 25 to 30 kg/m2). Excess body weight is one of the most important risk factors for all-cause morbidity and mortality. The likelihood of developing conditions such as type 2 diabetes, heart disease, cancer and osteoporosis of weight-bearing joints increases with body weight. The rapidly increasing world-wide incidence of obesity and its association with serious comorbid diseases means it is beginning to replace undernutrition and infectious diseases as the most significant contributor to ill health in the developed world.
It is now generally accepted that adipose tissue acts as an endocrine organ producing a number of biologically active peptides with an important role in the regulation of food intake, energy expenditure and a series of metabolic processes. Adipose tissue secretes a number of bioactive peptides collectively termed adipokines. Through their secretory function, adipocytes lie at the heart of a complex network capable of influencing several physiological processes. Dysregulation of adipokine production with alteration of adipocyte mass has been implicated in metabolic and cardiovascular complications of obesity. In obese individuals, excessive production of acylation-stimulating protein (ASP), TNFα, IL-6 or resistin deteriorates insulin action in muscles and liver, while increased angiotensinogen and PAI-1 secretion favors hypertension and impaired fibrinolysis. Leptin regulates energy balance and exerts an insulin-sensitizing effect. These beneficial effects are reduced in obesity due to leptin resistance. Adiponectin increases insulin action in muscles and liver and exerts an anti-atherogenic effect. Further, adiponectin is the only known adipokine whose circulating levels are decreased in the obese state. The thiazolidinedione anti-diabetic drugs increase plasma adiponectin, supporting the idea that adipokine-targeted pharmacology represents a promising therapeutic approach to control type 2 diabetes and cardiovascular diseases in obesity.
Metabolism of white adipose tissue is involved in the control of body fat content, especially visceral adipose tissue. Adipose tissue plays a central role in the control of energy homeostasis through the storage and turnover of triglycerides and through the secretion of factors that affect satiety and fuel utilization. Mitochondrial remodeling and increased energy expenditure in white fat may affect whole-body energy homeostasis and insulin sensitivity [Wilson-Fritch L, Nicoloro S, Chouinard M, Lazar M A, Chui P C, et al. 2004. Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 114: 1281-9].
Oxidative Stress—
Current consensus is that hyperglycemia results in the production of reactive oxygen (oxidative stress) and nitrogen species, which leads to oxidative myocardial injury. Alterations in myocardial structure and function occur in the late stage of diabetes. These chronic alterations are believed to result from acute cardiac responses to suddenly increased glucose levels at the early stage of diabetes. Oxidative stress, induced by reactive oxygen and nitrogen species derived from hyperglycemia, causes abnormal gene expression, altered signal transduction, and the activation of pathways leading to programmed myocardial cell deaths. The resulting myocardial cell loss thus plays a critical role in the development of diabetic cardiomyopathy.
Mitochondrial Uncoupling—
Controlling adiposity by targeted modulation of adipocyte mitochondrial membrane potential could offer an attractive alternative to current dietary approaches. It has recently been reported that forced uncoupling protein 1 (UCP1) expression in white adipocytes derived from a murine (3T3-L1) preadipocyte cell line reduced the total lipid accumulation by approximately 30% without affecting other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular ATP content to nutrient removal [Si Y, Palani S, Jayaraman A, Lee K. 2007. Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism. J Lipid Res 48: 826-36]. These results suggest that the targeting reduction in intracellular lipid of adipocytes by uncoupling mitochondrial membrane potential represents a feasible mechanism for identification of anti-obesity molecules. Nevertheless, the putative role of various mitochondrial protonophores in white fat cells in the control of adiposity remains to be clarified.
Thermogenesis—
Thermogenesis or uncoupling of mitochondrial membrane potential may be activated both indirectly and directly. Indirect activation occurs through β3AR and β3 agonists (β3AA). In the early 1980s, an “atypical” beta-adrenergic receptor was discovered and subsequently called β3AR. Further clinical testing will be necessary, using compounds with improved oral bioavailability and potency, to help assess the physiology of the β3AR in humans and its attractiveness as a potential therapeutic for the treatment of type 2 diabetes and obesity [de Souza C J, Burkey B F. 2001. Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesity drugs in humans. Curr Pharm Des 7: 1433-49].
Adaptive Thermogenesis—
Adaptive thermogenesis represents the decrease in energy expenditure (EE) beyond what could be predicted from the changes in fat mass or fat-free mass under conditions of standardized physical activity in response to a decrease in energy intake. Thus there exists the potential of adaptive thermogenesis to impede obesity treatment on a short- and long-term basis, at least in some individuals. In some cases, the adaptive decrease in thermogenesis was shown to be significantly related to a single cycle of body weight loss and regain, an increase in plasma organochlorine concentration following weight loss. This suggests that energy metabolism might be sensitive to stimuli of different physiological nature and that adaptive thermogenesis could be quantitatively more important than what is generally perceived by health professionals and nutrition specialists. However, from a clinical point of view, several issues remain to be investigated in order to more clearly identify adaptive thermogenesis determining factors and to develop strategies to cope with them. Along these lines, it is concluded that unsuccessful weight loss interventions and reduced body weight maintenance could be partly due, in some vulnerable individuals, to the adaptive thermogenesis, which is multicausal, quantitatively significant, and has the capacity to compensate for a given prescribed energy deficit, possibly going beyond any good compliance of some patients.
Additional approaches to increasing thermogenesis appear necessary to affect sustained weight loss in obese subjects. One of these approaches with demonstrated proof-of-concept in humans is direct, chemical stimulation of thermogenesis through chemical uncoupling of mitochondrial membrane potential using 2,4-dinitrophenol (DNP). Doubling metabolic rate by selectively and modestly uncoupling adipocyte thermogenesis should produce few adverse side-effects as this level of increase would only be equivalent to mild exercise. DNP is a lipid—soluble, weak acid that acts as a protonophore because it can cross membranes protonated, lose its proton and return as the anion, then reprotonate and repeat the cycle. In this way, it increases the basal proton conductance of mitochondria and uncouples oxidative phosphorylation. The overall result is a decrease in ATP formation for an equivalent amount of oxidation.
Inducible Nitric Oxide Synthase—
Obesity leading to insulin resistance is a major causative factor for type 2 diabetes and is associated with increased risk of cardiovascular disease. Despite intense investigation for a number of years, molecular mechanisms underlying insulin resistance remain to be determined. Recently, chronic inflammation has been highlighted as a culprit for obesity-induced insulin resistance. Nonetheless, upstream regulators and downstream effectors of chronic inflammation in insulin resistance remain unclarified. Inducible nitric oxide synthase (iNOS), a mediator of inflammation, has emerged as an important player in insulin resistance. Obesity is associated with increased iNOS expression in insulin-sensitive tissues in rodents and humans. Inhibition of iNOS ameliorates obesity-induced insulin resistance. However, molecular mechanisms by which iNOS mediates insulin resistance via nitric oxide (NO) biosynthesis remain largely unknown.
NO is a critically important signaling molecule, controlling a wide range of pathways and biological processes. Highly reactive nitric oxide mediates its function through reaction with different molecules directly or indirectly. One of these modifications is the S-nitrosylation of cysteine residues in proteins. S-nitrosylation is emerging as an important redox signaling mechanism and has been found to regulate a broad range of biologic, physiologic and cellular functions [Hausladen, A., and Stamler, J. S, Nitrosative stress. Methods Enzymol 1999, 300, 389-95].
Protein S-nitrosylation, a covalent attachment of NO moiety to thiol sulfhydryls, has emerged as a major mediator of a broad array of NO actions. S-nitrosylation is elevated in patients with type 2 diabetes, and increased S-nitrosylation of insulin signaling molecules, including insulin receptor, insulin receptor substrate-1, and Akt/PKB, has been shown in skeletal muscle of obese, diabetic mice. Akt/PKB is reversibly inactivated by S-nitrosylation. Based on these findings, S-nitrosylation has recently been proposed to play an important role in the pathogenesis of insulin resistance [Kaneki, M., Shimizu, N., Yamada, D., and Chang, K. Nitrosative stress and pathogenesis of insulin resistance. Antioxid Redox Signal 2007, 9, 319-29].
Moreover iNOS expression is increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorates depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance [Sugita, H., Fujimoto, M., Yasukawa, T., Shimizu, N., Sugita, M., Yasuhara, S., Martyn, J. A., and Kaneki, M. Inducible nitric-oxide synthase and NO donor induce insulin receptor substrate-1 degradation in skeletal muscle cells. J Biol Chem 2005, 280, 14203-11].
Improvements in the treatment of noncardiac complications from diabetes have resulted in heart disease becoming a leading cause of death in diabetic patients. Pathogenesis of diabetic cardiomyopathy (DCM) is a complicated and chronic process that is secondary to acute cardiac responses to diabetes. One of the acute responses is cardiac cell death that plays a critical role in the initiation and development of DCM. Besides hyperglycemia, inflammatory response in the diabetic heart is also a major cause for cardiac cell death. Diabetes or obesity often causes systemic and cardiac increases in tumor necrosis factor-alpha (TNFα), interleukin-18 and PAI-1. However, how these cytokines cause cardiac cell death remains unclear. It has been considered to relate to oxidative and/or nitrosative stress. Cardiac cell death is induced by the inflammatory cytokines that are increased in response to diabetes. Inflammatory cytokine-induced cardiac cell death is mediated by oxidative stress and is also the major initiator for DCM development [Wang, Y. H., and Cai, L. Diabetes/obesity-related inflammation, cardiac cell death and cardiomyopathy. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006, 31, 814-8].
AMP-Activated Protein Kinase—
The 5′-AMP-activated protein kinase (AMPK) functions as an intracellular fuel sensor that affects metabolism and gene expression in humans and rodents. AMPK has been described as an integrator of regulatory signals monitoring systemic and cellular energy status. Recently, it has been proposed that AMPK could provide a link in metabolic defects underlying progression to the metabolic syndrome. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. Rising AMP and falling ATP activate AMPK. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). The AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression (FIG. 3).
As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischemia playing a role in glucose transport and fatty acid oxidation. In liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production [Viollet, B., Mounier, R., Leclerc, J., Yazigi, A., Foretz, M., and Andreelli, F. Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders. Diabetes Metab 2007, 33, 395-402]. The net effect of AMPK activation is stimulation of hepatic fatty acid oxidation and ketogenesis, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipolysis and lipogenesis, stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulation of insulin secretion by pancreatic beta-cells.
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) represents a useful tool for identifying new target pathways and processes regulated by the AMPK protein kinase cascade. Incubation of rat hepatocytes with AICAR results in accumulation of the monophosphorylated derivative (5-aminoimidaz-ole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells.
AMPK also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischemia playing a role in glucose transport and fatty acid oxidation.
Additional approaches to affect sustained weight loss in obese subjects represent a critical need. Further, compounds or formulations that safely and effectively activate AMPK may function to stimulate hepatic fatty acid oxidation and ketogenesis, inhibit cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibit adipocyte lipolysis and lipogenesis, stimulate of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulate insulin secretion by pancreatic beta-cells.
Inflammatory Bowel Disease—
Each year inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, afflict more than one million people in the United States [Baumgart D C, Bernstein C N, Abbas Z, et al. IBD Around the world: Comparing the epidemiology, diagnosis, and treatment: Proceedings of the World Digestive Health Day 2010—Inflammatory bowel disease task force meeting. Inflamm Bowel Dis 2010]. The healthy gastrointestinal tract absorbs only the small molecules like those that are product of complete digestion. These molecules are the amino acids, simple sugars, fatty acids, vitamins, and minerals that the body requires for all the processes of life to function properly. The intestines, small intestine in particular, only allow these substances to enter the body due to the fact that the cells that make up the intestinal wall are tightly packed together. The intestines also contain special proteins called ‘carrier proteins’ that are responsible for binding to certain nutrients and transporting them through the intestinal wall and into the bloodstream.
Leaky Gut Syndrome (LGS) is common parlance for the disruption of the intestinal membrane integrity as a result of oxidative stressors or pro-inflammatory mediators, which compromise the ability of the intestinal wall to keep out large and undesirable molecules. Hence the name, as substances that are normally kept outside the body and within the intestines, are “leaking” across the intestinal wall and into the body as a whole. This happens when the spaces between the cells of the intestinal wall become enlarged for various reasons and allow larger, less digested particles and toxins to pass through—causing LGS. The body then recognizes these particles as foreign “invaders,” and the immune system attempts to fight them off—which can set the stage for various autoimmune disorders.
This disruption of intestinal membrane integrity is applicable to the pathognomic impacts of asthma, arthritis, food allergies, ulcers, Crohn's disease, ulcerative colitis, celiac disease, autoimmune diseases, alcoholism, chronic fatigue, joint pain, migraines, diarrhea, parasitic infections, dysbiosis, candidiasis, multiple sclerosis, diabetes, multiple sclerosis, vasculitis, Addison's disease, lupus, thyroiditis, and fibromyalgia.
Novel, supercritical CO2 extracts of N. sativa are described that contain from about 0.01 to about 39% (w/w) TQ and unexpectedly exhibit chemical and biological activities in vitro and clinically that differ both qualitatively and quantitatively from TQ, the putative active component of N. sativa seed extracts.
To date no process for supercritical extraction of N. sativa has been described that is useful for commercial quantities of ground seed. It is well known in the art, that changes in scale will profoundly affect the quality and quantity of the extract produced. The procedure described herein can be used in the extraction of commercial quantities of N. sativa seed in amounts greater than about 10 kg.